The inhibition of prostate-specific membrane antigen (PSMA) by tight-binding small-molecule inhibitors has not been fully exploited for chemotherapeutic or imaging strategies. Our long-term goal is to develop novel delivery mechanisms for either cytotoxic or imaging agents that capitalize on the potency and specific affinity of tight-binding inhibitors for cell-surface hydrolytic enzymes. The overall objective of this R21 application is to prove the concept that potent inhibitors of PMSA which bear a radionuclide-chelating motif that, when bound to technetium-99m (Tc-99m), will selectively tag prostate cancer cells for SPECT (single photon emission computed tomography). Our central hypothesis for the proposed work is that small-molecule inhibitors of PSMA outfitted with a metal-chelating motif can deliver radionuclides specifically to prostate cancer cells that express PSMA. The rationale for undertaking the proposed research is that, once we demonstrate that potent small-molecule inhibitors of PSMA bearing a radionuclide can be selectively delivered to PSMA-expressing cells, it will serve as a proof-of-concept for the development of novel therapeutic and imaging technology for prostate cancer in a subsequent R01 proposal. A library of selective PSMA inhibitors possessing a metal-chelating motif will first be generated using a modular synthetic approach. When loaded with Tb(lll) or Eu(lll), the fluorescent properties of these tethered metal-chelate inhibitors will allow for in vitro evaluation of these probes by fluorescence microscopy and flow cytometry. Lastly, the chelator-tethered inhibitors will be loaded with 99Tc to selectively deliver this imaging radionuclide specifically to PSMA-expressing cancer cells. The expected positive impact of these results is that a foundation for new diagnostic strategies for prostate cancer will be established. These accomplishments are important, because these novel inhibitors of PSMA can later modified to deliver other radionuclides suitable for therapeutic applications for prostate cancer. [unreadable] [unreadable] [unreadable]